Respuesta inmune específica frente a cmv y su relación con la protección frente a la infección para el diseño de una vacuna

Falta palabras claveA pesar de que la infección por CMV es generalmente asintomática en individuos inmunocompetentes, continúa siendo una de las principales causas de morbilidad y mortalidad tras el trasplante de órgano sólido. En un estudio previo realizado por nuestro grupo demostramos que la terapia anticipada en receptores de alto riesgo era segura y promovía la adquisición de una respuesta de células T específica de CMV que se asoció con un efecto terapéutico frente a la infección. Sin embargo, encontramos necesario analizar el papel deotros brazos de la respuesta inmune específica que podrían estar influyendo en este efecto protector, así como establecer puntos de corte para poder aplicarlo a la práctica clínica. Por su parte, el desarrollo de una vacuna preventiva frente a la infección por CMV ha ganado fuerza en los últimos años desde que fue propuesto como prioridad por el Instituto de Medicina Americano (IOM). Se han realizado distintos diseños de vacunación utilizando virus atenuados, DNA o proteínas recombinantes, pero hasta el momento ninguna de estas formulaciones han sido licenciadas para su uso en humanos. Es por ello que el conocimiento de los antígenos virales, su expresión, y su papel durante la infección serían buenas aproximaciones al desarrollo de una vacuna efectiva. El objetivo de este estudio fue en primer lugar definir parámetros de la respuesta humoral y celular específica de CMV relacionados con el control de la infección por CMV y enfermedad en una cohorte de pacientes TOS de alto riesgo de infección por CMV (D+/R-). En segundo lugar, analizar y comparar mediante Secuenciación de Nueva Generación (NGS) el perfil de expresión de los genes de CMV durante la infección de fibroblastos y células epiteliales para identificar los tránscritos virales más y diferencialmente expresados durante la infección de ambos tipos celulares, para identificar antígenos potenciales para el diseño de una vacuna. En la primera parte de esta tesis, observamos que aunque la respuesta celular T específica de CMV se asocia con la aclaración espontánea de la viremia de CMV, unpequeño porcentaje de pacientes con una respuesta celular T positiva están aún en riesgo de desarrollar enfermedad tardía por CMV. Además, demostramos que los pacientes con serología pretrasplante negativa para CMV desarrollan de novo una inmunidad humoral mediada por un amplio rango de anticuerpos neutralizantes. Aunque no encontramos relación entre los títulos de anticuerpos capaces de bloquear la infección de fibroblastos y la protección frente a la infección o la progresión clínica del paciente tras el trasplante, sí fuimos capaces de definir un punto de corte de títulos de anticuerpos neutralizantes de infección de epitelio (≥480) que se correlaciona con la disminución de la infección y la protección frente a la enfermedad por CMV. Nuestros resultados sugieren que la cuantificación de los títulos de anticuerpos neutralizantes de la infección de células epiteliales (título≥480), además de la respuesta celular T (CD8+ IFN-γ +≥0,25%), podría ser útil para distinguir entre pacientes que requieren una monitorización más cercana y aquellos que presentan un menor riesgo de desarrollar enfermedad por CMV. De esta forma, la combinación de la monitorización virológica e inmunológica de los pacientes podría optimizar la administración de la terapia anticipada en pacientes de alto riesgo de infección por CMV. Sin embargo, será necesario llevar a cabo estudios prospectivos con cohortes de pacientes más numerosas para confirmar los resultados presentados. La segunda parte de la tesis consiste en el estudio por RNA-Seq de los tránscritos virales durante la infección de fibroblastos y células epiteliales. Observamos que una gran cantidad de transcritos virales diferían entre ambos tipos celulares infectados por CMV, fibroblastos vs. células epiteliales. En este trabajo se postuló que los genes más expresados durante la infección por CMV con un papel importante en la estimulación de una respuesta inmune específica frente a CMV podrían ser buenos candidatos para el desarrollo de una vacuna frente a CMV. Por ello, proponemos que UL36 y US3, transcritos altamente expresados podrían ser de especial interés por su capacidad de activar tanto una respuesta de células T CD4+ como CD8+. Además, debido a que las células epiteliales son la primera barrera biológica encontrada por CMV y que la respuesta humoral específica de CMV tras la infección natural está dirigida especialmente a bloquear la infección de células epiteliales, US18, detectado en nuestro estudio como el mayor MAT durante las tres fases de infección por CMV de células epiteliales, podría ser un buen candidato a vacuna ya que además presenta la capacidad de activar la respuesta celular mediada por células T CD4+

Infección por Citomegalovirus en Receptores de Trasplante de Órgano Sólido y efecto terapéutico de la adquisición de respuesta inmune específica

Motivación: A pesar de las mejoras de tratamiento, la infección por CMV es la principal causa de morbimortalidad en los pacientes con  trasplante de órgano sólido (TOS) con una incidencia de enfermedad tardía (>3 meses post-trasplante) por CMV que varía entre 6-30% (1).  La respuesta inmune mediada por células T capaces de secretar IFN-gamma tras la estimulación con CMV constituye la principal defensa frente a la infección viral (2) y juega un papel crucial en el control de las infecciones post-trasplante. Los pacientes seronegativos para CMV que reciben un trasplante de un donante seropositivo (D+/R-), son considerados de alto riesgo de desarrollar infección por CMV tras el trasplante ya que no presentan inmunidad previa.  El tratamiento anticipado con ganciclovir frente a CMV iniciado cuando la carga viral  alcanza un umbral determinado (3), no está recomendado en pacientes de alto riesgo, los cuales reciben profilaxis continua durante 100 días post-trasplante. Nuestra hipótesis fue que la administración de tratamiento anticipado en pacientes D+/R- podría permitir la exposición controlada al CMV que promoviera el desarrollo de inmunidad específica.Métodos: Se incluyeron pacientes D+/R- y se recogieron muestras de seguimiento durante 18 meses tras el trasplante. Se determinó la carga viral de CMV por PCR en tiempo real. Para determinar la respuesta inmune de células T la sangre completa fue estimulada con péptidos específicos de CMV (pp65 y IE-1), se llevó a cabo la tinción con anticuerpos de moléculas de superficie (CD8, CD4, CD69), y citoquinas intracelulares implicadas en la respuesta celular (IL2,IL4 e IFN-g), y se análizó mediante citometría de flujo. Además se está determinando los títulos de anticuerpos neutralizantes para bloquear la infección de células epiteliales y fibroblastos.Resultados: Los 21 pacientes estudiados desarrollaron episodios de replicación con una mediana de 4 semanas tras el trasplante, y una respuesta inmune específica con una mediana de 12 semanas. La incidencia de los episodios de replicación estuvo inversamente relacionada con la adquisición de la respuesta inmune. Tras la adquisición de la respuesta inmune todos los episodios de replicación fueron aclarados sin necesidad de tratamiento, ninguno de ellos desarrolló enfermedad tardía por CMV ni episodios de rechazo.Conclusiones: El tratamiento anticipado es eficaz para prevenir la infección por CMV en paciente D+/R- y promueve la respuesta inmune específica protectora

Validation of STAT1 variants found in patients with primary immunodeficiencies and evaluation of the effect of JAK inhibition using an in vitro model

Regulation of cellular responses to interferons, cytokines, growth factors and hormones is mediated by signal transducer and activator of transcription (STAT) proteins. In the immune system binding of a cytokine (e.g. IFN) to the corresponding surface receptor, Janus kinase (JAK) molecules are phosphorylated, resulting in the docking and phosphorylation of the associated STAT proteins. The STATs will form homo or heterodimers and translocate to regulate transcription of pro-inflammatory target genes. Mutations in STAT1 are known to result in immunodeficiency and/or immune dysregulation syndromes. In this project, the functional impact of variants in the STAT1 gain-of-function gene (STAT1 GOF) will be analyzed on a protein level. The effect of a directed treatment approach targeting the JAK-STAT pathway (JAK inhibitors) will be evaluated in an in vitro model. Freshly isolated PBMCs or whole blood samples from patients and healthy controls were obtained. The cells were stimulated with IFNg and the treated with the JAK inhibitor Ruxolitinib. Extra and intracellular staining with anti-human fluorochrome conjugated antibodies was performed in order to determine the expression of STAT1 and pSTAT1 on monocytes by means of flow cytometry. Two pediatric patients and one related adult patient were studied and the pathogenicity of the variants was confirmed as STAT1 and pSTAT1 levels in the patients at baseline as well as after IFNg stimulation were markedly increased, when compared with healthy controls. The in vitro administration of different concentrations of the JAK inhibitor Ruxolitinib resulted in the normalization of pSTAT1 levels in the cells obtained from patients with STAT1 GOF mutations. Patients with STAT1 GOF mutations show a severe clinical phenotype with recurrent bacterial and fungal infections. Currently no specific treatment options are available. However recent case reports suggested the benefit of JAK inhibition. Therefore we studied the effect of this drug using primary cells in an in vitro model on a molecular level. Importantly two patients have been started on this medication and have achieved a significant clinical response. We are currently evaluating the capacity of our protocol to identify patients with alterations of the JAK-STAT pathway eligible for this targeted treatment approach

Immune dysregulation in children with Down Syndrome and Janus Kinase inhibition as targeted therapy.

Down Syndrome (DS), also known as Trisomy 21, is a genetic disorder that results from partial or complete third copy of chromosome 21. It is one of the most common genetic disorders worldwide affecting 6 million individuals. It is the leading cause of lifelong intellectual disability and is often associated with other clinical alterations [1], including cardiac and gastrointestinal anatomic defects, an increased incidence of Alzheimer's disease as well as a heightened vulnerability to infections.DS has shown to be associated with inflammatory and autoimmune processes such as periodontitis, alopecia, dermatitis among others [2]. The fact that 4/6 Interferon (IFN) receptors are localized on chromosome 21 is currently an appealing hypotheses to explain the observed IFN hypersensitivity in DS population. A subset of DS individuals shares clinical manifestations with patients with Signal-Transducer and Activator of Transcription 1 gain-of-function (STAT1 GOF) patients, who also present IFN hypersensitivity and increased levels of STAT1 and its phosphorylated form (pSTAT1) [3]. Janus Kinase (JAK) inhibition has been a successful treatment for STAT1 GOF patients. However, its potential and effectiveness in Down Syndrome has not been explored yet. We here propose Baricitinib as a targeted therapeutic approach for a selected subgroup of DS patients with IFN hypersensitivity and clinical manifestations similar to the ones seen in patients with STAT1 GOF. Whole blood (WB) obtained from STAT1 GOF patients, DS individuals and healthy controls (HC) was stimulated with IFN αlpha/gamma in the presence of Baricitinib. Flow cytometry was performed using specific antibodies against CD14 (monocytes), CD3, STAT1 and pSTAT1. In parallel, PBMCs were stimulated with IFN alpha/gamma , and transcription levels of STAT1, CxCL10, SOCS1 and PD-L1 were evaluated by RT-PCR. According to previous studies [3], we found in DS individuals an increased gene dosage of IFNaR1 and IFNaR2. Furthermore, similar to STAT1 GOF patients some DS individuals present increased levels of STAT1 and pSTAT1 after stimulation compared to HC in CD14 and CD3 T cells. We also observed that ex vivo JAK inhibition was effective reducing this IFN hypersensitivity in DS cells similar to STAT1 GOF patients’ cells. Taken together, Baricitinib a JAK1/2 inhibitor might represent a promising targeted therapy for a subgroup of DS with inflammatory and/or infectious manifestations

Searching for predictors of the variability of impacts caused by non-native trees on regulating ecosystem services worldwide

Humans have introduced non-native trees (NNT) all over the world to take advantage of the plethora of benefits they provide. However, depending on the context, NNT may present a diverse range of effects on ecosystem services (ES), from benefits to drawbacks, which may hinder the development of policies for these species. Unfortunately, the attempts so far to understand the impacts of NNT on ES only explained a low proportion of their variation. Here we analyze the variation in impacts of NNT on regulating ecosystem services (RES) by using a global database, which covers the effect size of multiple NNT species on six RES (climate regulation, soil erosion regulation, soil fertility, soil formation, hydrological cycle regulation, and fire protection). We used a wide range of predictors to account for the context-dependency of impacts distributed in five groups: the RES type, functional traits of both the NNT and the dominant NT of the recipient ecosystem, phylogenetic and functional distances between NNT and NT, climatic context, and human population characteristics. Using boosted regression trees and regression trees, we found that the most influential predictors of NNT impacts on RES were annual mean temperatures and precipitation seasonality, followed by the type of RES, human population density, and NNT height. In regions with warm temperatures and low seasonality, NNT tended to increase RES. NNT impacts were greater in densely populated regions. Smaller NNT exerted greater positive impacts on climate regulation and soil erosion regulation in tropical regions than in other climates. We highlight that benign climates and high population density exacerbate the effects of NNT on RES, and that soil fertility is the most consistently affected RES. Knowledge of the factors that modulate NNT impacts can help to predict their potential effects on RES in different parts of the world and at various environmental setting

Ex vivo efect of JAK inhibition on JAK‑STAT1 pathway hyperactivation in patients with dominant‑negative STAT3 mutations

Purpose STAT1 gain-of-function (GOF) and dominant-negative (DN) STAT3 syndromes share clinical manifestations includ ing infectious and infammatory manifestations. Targeted treatment with Janus-kinase (JAK) inhibitors shows promising results in treating STAT1 GOF-associated symptoms while management of DN STAT3 patients has been largely supportive. We here assessed the impact of ruxolitinib on the JAK-STAT1/3 pathway in DN STAT3 patients’ cells. Methods Using fow cytometry, immunoblot, qPCR, and ELISA techniques, we examined the levels of basal STAT1 and phosphorylated STAT1 (pSTAT1) of cells obtained from DN STAT3, STAT1 GOF patients, and healthy donors following stimulation with type I/II interferons (IFNs) or interleukin (IL)-6. We also describe the impact of ruxolitinib on cytokine induced STAT1 signaling in these patients. Results DN STAT3 and STAT1 GOF resulted in a similar phenotype characterized by increased STAT1 and pSTAT1 levels in response to IFNα (CD3+ cells) and IFNγ (CD14+ monocytes). STAT1-downstream gene expression and C-X-C motif chemokine 10 secretion were higher in most DN STAT3 patients upon stimulation compared to healthy controls. Ex vivo treatment with the JAK1/2-inhibitor ruxolitinib reduced cytokine responsiveness and normalized STAT1 phosphorylation in DN STAT3 and STAT1 GOF patient’ cells. In addition, ex vivo treatment was efective in modulating STAT1 downstream signaling in DN STAT3 patients. Conclusion In the absence of efective targeted treatment options for AD-HIES at present, modulation of the JAK/STAT1 pathway with JAK inhibitors may be further explored particularly in those AD-HIES patients with autoimmune and/or autoinfammatory manifestations

A bivalent live-attenuated vaccine for the prevention of equine influenza virus

Vaccination remains the most effective approach for preventing and controlling equine influenza virus (EIV) in horses. However, the ongoing evolution of EIV has increased the genetic and antigenic differences between currently available vaccines and circulating strains, resulting in suboptimal vaccine efficacy. As recommended by the World Organization for Animal Health (OIE), the inclusion of representative strains from clade 1 and clade 2 Florida sublineages of EIV in vaccines may maximize the protection against presently circulating viral strains. In this study, we used reverse genetics technologies to generate a bivalent EIV live-attenuated influenza vaccine (LAIV). We combined our previously described clade 1 EIV LAIV A/equine/Ohio/2003 H3N8 (Ohio/03 LAIV) with a newly generated clade 2 EIV LAIV that contains the six internal genes of Ohio/03 LAIV and the HA and NA of A/equine/Richmond/1/2007 H3N8 (Rich/07 LAIV). The safety profile, immunogenicity, and protection efficacy of this bivalent EIV LAIV was tested in the natural host, horses. Vaccination of horses with the bivalent EIV LAIV, following a prime-boost regimen, was safe and able to confer protection against challenge with clade 1 (A/equine/Kentucky/2014 H3N8) and clade 2 (A/equine/Richmond/2007) wild-type (WT) EIVs, as evidenced by a reduction of clinical signs, fever, and virus excretion. This is the first description of a bivalent LAIV for the prevention of EIV in horses that follows OIE recommendations. In addition, since our bivalent EIV LAIV is based on the use of reverse genetics approaches, our results demonstrate the feasibility of using the backbone of clade 1 Ohio/03 LAIV as a master donor virus (MDV) for the production and rapid update of LAIVs for the control and protection against other EIV strains of epidemiological relevance to horses

Double Trouble: a patient with STAT1 GOF and Down Syndrome

Multiple biological processes are regulated by different cytokines through signal transducer and activator of transcription (STAT) factors. The type I interferons (IFN) activate the JAK-STAT pathway, that induces the tyrosine phosphorylation of STAT1 (pSTAT1) and its binding to the DNA to activate transcription of STAT-dependent genes. Gain of function (GOF) mutations in STAT1 result in hyperactivation of STAT1 and upregulation of downstream genes. Patients with STAT1 GOF present a wide spectrum of clinical manifestations characterized by a reduction of circulating Th17 cells and high susceptibility to chronic mucocutaneous candidiasis (CMC). A cluster of six interferon receptors (IFN-R) genes is required for the detection of the different types of IFN. Four of the six IFN-R genes are located on the human chromosome 21. Down syndrome (DS), or trisomy 21, is one of the most known diseases causing intellectual disability. In addition, DS patients are more frequent to present autoimmune phenotypes and fungal infections like CMC (56-76%). In common with STAT1 GOF, DS patients could suffer from similar autoimmune disorders due to hyperresponsiveness to IFNs. In this project, the functional impact of both diseases will be analysed in a patient with STAT1 GOF and DS. Freshly isolated PBMCs or whole blood were obtained from a STAT1 GOF and DS patient, her mother and healthy controls. Flow cytometry analysis was conducted on whole blood treated in vitro with IFNα, IFNγ and IL6. Extra and intracellular staining with different antibodies specific for monocytes, STAT1 and pSTAT1 was performed for the study of STAT1 phosphorylation. PBMCs were stimulated with cytokines and qPCR was conducted in order to measure the gene transcription of STAT1, CxCL10, SOCS1, SOCS3, PIAS1 and PIAS3. The study of the pathogenicity of both diseases, STAT1 and DS, were carried out in a pediatric patient with STAT1 GOF and DS that presented severe disease and her mother who shares the same mutation in STAT1 and mild symptoms. The analysis of the STAT1 and pSTAT1 levels, before and after stimulation, indicated an elevated level in both patients compared with healthy controls. However, the levels of total STAT1 in the pediatric patient showed a greater increase in comparison to her mother. These results could explain part of the severe phenotype in the pediatric patient. Importantly this patient has been receiving treatment for a year which has markedly improved her quality of life

Biallelic TRAF3IP2 variants causing chronic mucocutaneous candidiasis in a child harboring a STAT1 variant

[Background] Inherited chronic mucocutaneous candidiasis (CMC) is often caused by inborn errors of immunity, impairing the response to, or the production of IL-17A and IL-17F. About half of the cases carry STAT1 gain-of-function (GOF) mutations. Only few patients have been reported with mutations of TRAF3IP2, a gene encoding the adaptor ACT1 essential for IL-17 receptor(R) signaling. We investigated a 10-year-old girl with CMC, carrying a heterozygous variant of STAT1 and compound heterozygous variants of TRAF3IP2.[Methods] By flow cytometry, STAT1 levels and phosphorylation (CD14+) as well as IL-17A, IL-22, IFN-γ, and IL-4 production (memory CD4+ T cells) were determined. ACT1 expression and binding to IL-17RA were assessed by Western blot and co-immunoprecipitation in HEK-293T cells transfected with plasmids encoding wild-type or mutant HA-tagged ACT1 and Flag-IL-17RA. We evaluated IL-17A responses by measuring luciferase induction under a NF-κB-driven reporter system in HEK-293T cells and Gro-α secretion in fibroblasts. [Results] A STAT1 variant (c.1363G>A/p.V455I) was identified by next-generation sequencing and classified as likely non-pathogenic as functional testing revealed normal STAT1 expression and phosphorylation upon IFN-γ. We also found compound heterozygous variants (c.1325A>G/p.D451G and c.1335delA/p.K454fs11*) of TRAF3IP2. By overexpression, despite normal protein expression, and impaired (K454fs11*) or normal (D451G) interaction with IL-17RA, both mutant alleles resulted in impaired NF-κB activation. Patient's fibroblasts displayed abolished GRO-α secretion upon IL-17A stimulation. Finally, ex vivo CD4+ T cells showed increased IL-17A, IL-22, and IL-4 and normal low IFN-γ expression upon stimulation. [Conclusion] We identify novel compound heterozygous variants of TRAFP3IP2 causing autosomal recessive ACT1 deficiency in a child with CMC and provide a review of the current literature